Molecular characterization of extended-spectrum beta-lactamases (ESBLs) produced by clinical isolates of Acinetobacter baumannii in Saudi Arabia

Acinetobacter baumannii is a common opportunistic pathogen that causes major nosocomial infections in hospitals. In this study, we hypothesized a high prevalence of A. baumanni ESBL (extended-spectrum beta-lactamase) among all collected isolates. A. baumannii isolates (n = 107) from ICU (Intensive care unit) of local hospitals in Makkah were phenotypically and genotypically characterized. The identity and antibiotic susceptibility of A. baumannii strains were determined using the Vitek-2 system. The identified ESBL producers were further analyzed by PCR and sequencing followed by MLST typing. blaTEM, blaSHV, and the blaCTX-M-group genes 1, 2, 8, 9, and 25 were investigated. Furthermore, blaOXA51-like and blaOXA23-like genes were also examined in the carbapenem-resistant A. baumannii isolates. Our data indicated a high prevalence of A. baumannii ESBL producers among the collected strains. Of the 107 A. baumannii isolates, 94 % were found to be resistant to cefepime and ceftazidime, and aztreonam using the Vitek 2 system. The genes detected encoded TEM, OXA-51-like and OXA-23-like enzymes, and CTX-M-group proteins 1, 2, 8, 9, and 25. MLST typing identified eight sequence type (ST) groups. The most dominant STs were ST195 and ST557 and all of them belong to worldwide clonal complex (CC) 2. This study has shown that there is a high prevalence of antimicrobial resistance in A. baumannii. The diversity of STs may suggest that new ESBL strains are constantly emerging. The molecular diversity of the ESBL genes in A. baumannii may have contributed to the increased antimicrobial resistance among all isolates.


Background
Acinetobacter baumannii is an opportunistic and rapidly emerging pathogen. It is an important agent of nosocomial infections worldwide, such as urinary tract infections, septicemia, pneumonia, burns, meningitis, and wound infections in hospitals, due to its remarkable propensity to rapidly acquire resistance determinants to a wide range of antibacterial agents [1][2][3][4]. Many studies have documented high rates of multidrug-resistance (MDR) in A. baumannii [4][5][6]. The development of resistance to the third-generation cephalosporins was a major breakthrough in the fight against MDR strains. However, due to the frequent use of these agents, new plasmids encoding β-lactamase capable of hydrolyzing extended-spectrum cephalosporins were first reported in 1983 [7,8]. These extended-spectrum β-lactamases (ESBLs) are mutant, plasmid-mediated, and produced by gram negative bacilli that mediate resistance to penicillin, cephalosporins, and Open Access *Correspondence: eyamani@kacst.edu.sa 1 Molecular Bacteriology, National Center for Biotechnology, King Abdulaziz City for Science and Technology, P.O. Box 6086, Riyadh 11442, Saudi Arabia Full list of author information is available at the end of the article monobactams [9]. These ESBLs are commonly recognized in Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii and are found worldwide [10]. The majority of ESBLs are members of either the TEM, SHV, or CTX-M (class A) families based on the Ambler molecular classification of β-lactamase genes [11,12]. One of the major genes of ESBL family is the CTX-M, which is divided into five phylogenetic groups based on amino acid sequence identity: the CTX-M-1 group, the CTX-M-2 group, the CTX-M-8 group, the CTX-M-9 group, and the CTX-M-25 group. The presence and prevalence of these different groups are variable depending on the geographical locale [13,14]. In Saudi Arabia, the high prevalence of ESBL A. baumannii was reported in several studies [15][16][17]. The PCR technology is widely used technique to screen for ESBL in modern hospitals. A specific multiplex PCR assay has been optimized to screen for multiple ESBL genes to facilitate and monitoring the spread and emergence of ESBL-producing bacteria [18]. The epidemiologic characterization of A. baumannii by multilocus sequence typing (MLST) is a highly used method and has been applied successfully [19]. With reports on the high prevalence of ESBL production in members of A. baumannii globally and a paucity of information specifically regarding the emergence of ESBL A. baumannii in major Saudi general hospitals in Makkah, this study reports the analysis of the antibiotic susceptibility profiles and molecular characterization of 107 A. baumannii ESBL producers isolated from ICU ward based on the phenotypic and genotypic approach. Understanding the molecular nature of the spread of A. baumannii in local hospitals is important, especially in hospitals that admit thousands of local and foreign people during their holy journey to Makkah. This work may enhance our understanding of the extent of the epidemiologic re-emergence of this bacterium. The genes that were investigated from A. baumannii isolates by PCR were bla TEM , bla SHV , and the bla CTX-Mgroup genes 1, 2, 8, 9, and 25. Furthermore, bla OXA51-like and bla OXA23-like enzymes were examined in carbapenemresistant A. baumannii. This work may partially contribute to the global effort to map the molecular signature of A. baumannii.

Study design
A total of 107 bacterial isolates were collected from different ICU patients from clinical labs at local general hospitals in Makkah during 2 years from 2012 to 2014. Samples were subjected to a conventional microbiology analysis, phenotyping, and genotyping characterizations at the national center for biotechnology, KACST. The nature of the samples were blood, and skin wound infections predominantly.

Species identification and antimicrobials susceptibilities
Bacterial identities were confirmed using the Vitek 2 system (GN ID Card, bioMérieux, Craponne, France) and PCR. Antibiotic susceptibility testing was conducted according to the manufacturer's recommendations (gram negative antimicrobial susceptibility testing (AST) cards, bioMérieux, Craponne, France). The extraction of genomic DNA was performed using QIAGEN kits (

Detection of ESBL and carbapenem genes by PCR
All of the positive ESBL isolates according to phenotypic assays (n = 100) were further confirmed by PCR and sequencing. The genes investigated in this study were the bla TEM , bla SHV , and bla CTX-M-group genes 1, 2, 8, 9, and 25. Furthermore, bla OXA51-like and bla OXA23like enzymes were tested for carbapenem-resistant A. baumannii. The gDNA was extracted using a QIAamp Genomic DNA kit (QIAGEN, Venlo, Netherlands) and used for PCR directly, or overnight cultures were boiled at 95 °C for 10 min to produce a bacterial gDNA/plasmid lysate that was diluted 1:10 with ddH 2 O before it was used for PCR. PCR amplification was performed with either 1 µl of pure gDNA or 10 µl of gDNA/plasmid lysate as a template. Final reactions of 25 μl of illustra PuReTaq Ready-To-Go PCR beads (GE Health Biosciences, USA) were used in the PCR reaction according to the manufacturer's recommendations. The reactions were set up as follows: 10-22 μl of nucleasefree water (Promega) depending on the DNA templates being used; 2 µl of 10 pmol of each bla TEM , bla SHV , and bla CTX-M-group genes 1, 2, 8, 9, and 25; bla OXA51-like and bla OXA23-like forward and reverse primers (Eurofins MWG Operon, Germany); and 1-10 µl of DNA template or bacterial lysate were used (Table 1) [18]. The cycling conditions of the PCR are illustrated in Table 1. All of the amplicons were size fractionated using 1 % agarose gel electrophoresis and visualized under ultraviolet illumination using the Gel Doc EZ system (Bio-Rad, Hercules, CA, USA).

Amplification and sequencing of the 16S rRNA gene
Amplification and sequencing of 16S rRNA were performed to confirm the identity of A. baumannii used in this study [20]. In the PCR amplification, each reaction contained 25 μl of illustra PuReTaq Ready-To-Go PCR beads (GE Health Biosciences, USA). The reaction was set up as follows: 22 μl of nuclease-free water (Promega), 2 µl of 10 pmol of each forward and reverse primer (Eurofins MWG Operon, Germany) were used (Table 1) [18]. Exactly 1 μl of 100 ng/µl DNA template was added to the beads. The amplification conditions are highlighted in Table 1. The amplification products were subjected to gel electrophoresis in 1 % agarose followed by ethidium bromide staining and were visualized under ultraviolet illumination using the Gel Doc EZ system (Bio-Rad, Hercules, CA, USA). Sense and anti-sense strands of PCR amplicons were purified and sequenced in an ABI 3130 Genetic Analyzer (Life Technologies, Carlsbad, CA, USA) using ABI BigDye terminator cycle sequencing ready reaction kit chemistry according to the manufacturer's recommendations. Following sequencing, the data were identified using a basic local alignment search tool BLAST-n (http://www. ncbi.nlm.nih.gov/BLAST) or RDP database [21]. The identification of A. baumannii using the 16S rRNA was unequivocal. Therefore, there was no need to use additional confirmatory targets such as rpoB and gyrB genes [22].

Multilocus sequence typing (MLST)
The Acinetobacter baumannii complex MLST typing was performed by utilizing seven house-keeping genes: Citrate synthase (gltA), DNA gyrase subunit B (gyrB), Glucose dehydrogenase B (gdhB), Homologous recombination factor (recA), 60-kDa chaperonin (cpn60), Glucose-6-phosphate isomerase (gpi), RNA polymerase sigma factor (rpoD). The primers used for amplification and sequencing are illustrated in Table 2 [19]. The PCR amplifications were completed in a MasterCycler nexus (Eppendorf, Hamburg, Germany) with the following conditions: 35 cycles of initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min and 4 min final extension at 72 °C. The PCR products were directly verified by 1 % agarose gel electrophoresis before they were purified from the reaction mixture for sequencing. Bidirectional sequencing was performed for each isolate. Different allele sequences were assigned for each locus with an arbitrary allele number for identification. Each bacterial isolate was characterized by a pattern of numbers defining its sequence type (ST). The sequences of the seven housekeeping  [23]. The allelic profile similarities were produced by BioNumerics version (7) created by Applied Maths NV. Available from (http:// www.applied-maths.com).

Ethics statement
Ethical approval and consent were not required for this project because no human nor animal subjects were used.

16S rRNA identification and the detection of ESBL and carbapenemase genes
The 16S rRNA sequencing of all isolates (n = 107) generated a high score (≥97 % in total) of A. baumannii identity using the BLAST and Ribosomal Database Project  (Table 3; Fig. 2). The sequencing analysis of all of the genes showed approximately 90 % sequence similarity to the submitted sequences that are related to the genes deposited in GenBank.

Multilocus sequence typing analysis
MLST and sequence-based typing of ESBL and carbapenemase isolates were performed to analyze the genetic relationship of all of the isolates. The MLST anaylsis contains 97 isolates of 102. Five isolates were not typable due to low quality traces files and were not assigned STs but were included in the dendrogram. The MLST analysis allowed us to group the A. baumannii isolates into eight STs (Figs. 3, 4). MLST typing showed that the most dominant sequence type was ST195 (n = 69), followed by ST557 (n = 6), ST 208 (n = 4), ST499 (n = 2), ST218 (n = 2), ST231 (n = 1), ST222 (n = 1), and ST286 (n = 2). All of STs except ST 231 belonge to clonal complexity 2 (CC2) and lineage clone 2. The tree (Fig. 3) is based on the nucleotide sequence of at least 6 or 7 housekeeping genes. The analysis was based on data sets that include all STs in the Pasteur MLST databases of A. baumannii (http://pubmlst.org/abaumannii/).

Discussion
In this study, we detected and characterized the phenotypic and genotypic nature of ESBL producers in A. baumannii, which were isolated from general hospitals in Makkah, Saudi Arabia. At least 107 A. baumannii isolates were characterized by the Vitek-2 system and PCRsequencing followed by MLST typing. Our data indicated a high prevalence of A. baumannii ESBL producers among the collected isolates. A remarkable outcome of this study was the large number of antibiotic resistance genes found in these isolates. Ninety-four percent of A. baumannii isolates were found to have three major resistant determinants. We speculate that if more drugresistant genes were screened, we would have found panresistant A. baumannii isolates. CTX-M β-lactamases produced by A.baumannii strains is plasmid-mediated hence the wide spread and long time survival in hospitals. The CTX-M gene activity conferring resistance to cefotaxime and ceftazidime. We detected CTX-M group 1, 2, 8, 9, 25 in our current study (81 %). The high rate of prevalence of CTX-M resistance in gram negative bacteria may be influenced by mobile genetic elements around these genes which include transposon, insertion sequences (IS) and integrons [25]. Consistent with our study, all gram negative CTX-M producing bacteria are often associated with other families of other β-lactamases resistance causing multi-drug resistance phenomena. The high prevalence rate around the world of CTX-M makes it a predominant drug resistant gene in gram negative bacteria [26].
We studied the dynamic spread of A.baumannii in our population by MLST. The discriminatory power of the MLST system is comparable to other techniques such as pulsed field gel electrophoresis (PFGE). Yet, MLST provides a quick and easy method to study the epidemiology of ESBL-producing bacteria and to monitor the international emergence of multidrug resistant bacteria. Consistent with other studies that used MLST in the epidemiologic characterization of clinically important bacterial pathogens such as A.baumannii, Streptococcus pneumoniae, Streptococcus pyogenes, Neisseria meningitidis, Campylobacter jejuni, Staphylococcus aureus, Enterococcus faecium, Haemophilus influenza, and Vibrio cholera, our study has detected different allelic diversity (STs) which belongs to clonal complex (CC)2 which is globally distributed in Europe, Asia, Africa, Australia, USA, South America [19,[27][28][29].
The drug of choice to treat nosocomial infection caused by A.baumannii is the carbapenems. However, there is an increasing rate of carbapenem-resistant A.baumannii around the world caused by OXA23-like enzume or OXA51-like enzyme acitivies [30]. The first OXA23like enzyme with carbapenem-activity to A. baumannii was isolated and characterized in Scotland in 1985. This   [31]. This may explain the high prevalence of carbapenemase-producing A. baumannii in hospitals around the world. The other gene cluster in the OXA family is the bla OXA-51 -like gene which is chromosomally encoded and naturally occurs in A. baumannii. The functional product of this gene delivers carbapenemase resistance to meropenem and imipenem; its role in carbapenem resistance may be influenced by the presence of ISAba1. PCR mapping studies have found that the absence of this sequence upstream of blaOXA-51-like gene may contribute to a minimal effect on carbapenem susceptibility [32][33][34][35][36]. A recent study in the Gulf Countries Council (GCC) [37], namely, Saudi Arabia, the United Arab Emirates, Oman, Qatar, Bahrain, and Kuwait, suggested a high prevalence of carbapenemase resistance in A. baumannii, Escherichia coli and Klebsiella pneumonia. A. baumannii (n = 117) was studied as clusters in seven different sequence types: ST195, ST208, ST229, ST436, ST450, ST452 and ST499. Three of these sequences were identified in our study, including ST195, ST499, and ST208, which may suggest the circulation of these three STs in GCC countries [17,37]. The circulation of the STs within GCC may be due to the closeness of these countries to each other. Recent reports have been accumulating from Saudi Arabia due to the wide and rapid spread of carbapenem-resistant gram negative bacteria isolated from local hospitals specially during high season [38][39][40].
The high level of detection of ESBL and carbapenemase resistance among local isolates may suggest an increasing incidence rate of infection with ESBL-producing A. baumannii. Such high rates of ESBL-producing bacteria may impose a burden on routine clinical practice, especially for patients with chronic diseases and immunocompromised patients. Although national surveillance data are lacking, outbreaks of infection due to ESBL-producing A. baumannii have been reported by many hospitals within the Kingdom of Saudi Arabia. The true prevalence of ESBL producers is not known and is likely underestimated because of the difficulties encountered in their detection by most local hospitals. However, it is clear that ESBL-producing bacteria are distributed worldwide and their prevalence is increasing [2,6,41,42]. Therefore, periodic screening of ESBL-producing A. baumannii during the high hospital visitation season is recommended in all local hospitals to establish national surveillance data archives of the level of spread of ESBL producers.

Conclusions
In this study, we randomly surveyed and characterized ESBL-producing A. baumannii from ICU of local hospitals in Makkah city, Saudi Arabia. Our data indicated a high prevalence of A. baumannii ESBL producers among the collected isolates. Based on MLST typing, we have evidence of eight STs groups in our isolates. The epidemiologic diversity of these isolates may suggest that new ESBL strains are constantly emerging. The molecular diversity of the ESBL genes in A. baumannii may have contributed to the increased antimicrobial resistance among all isolates. Therefore, periodic screening of ESBL-producing A. baumannii during the high hospital visitation season is recommended in all local hospitals.